USPTO Patents Application 10687850

PCX

WORLD INTELLECTUAL PROPERTY ORGANIZATIOK
mtemational Bureau

INTERNATIONAL AMPLICATION PUBUSHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) InternatiooBl Patent Classification ^ :
GOIN

A2

(II) International Publication Number:
(43) International Publication Date:

WO 96/13707

9 May 1996 (09.05.96)

(21) International Ap|;Sication Number: PCTAJS95/I2550

(22) International Filing Date: 20 October 1 99S (20.10.95)

(30) Priority Data:
08/326.788

20 October 1994 (20.10.94)

US

(71)(72) Applicant and Inventon CONNOLLY, James [US/US);
6131 West 80th Street. Indianapolis. IN 46278 (US).

(74) Agent: LFTZINGER. JcrroW, J.; Suite 600, 4445 Lake Foicsi
Drive. Cincinnati. OH 45242 (US).

(81) Designated States: CA. JP. European patent (AT. BE. CH. DE,
DK. ES, FR. GB. GR, IE. IT. LU. MC, NL. PT, SE).

Published

Without international search report and to be republished
upon receipt cf that report.

(54) Tiae: APPARATUS AND ^4ETHOD FOR DETERMINING SUBSTANCES CONTAINED IN A BODY FLUID

(57) Abstract

An apparatus for the q>toeIectrontc evaluation of test paper strips for use in the detection of ceitain analytes in Mood or other body
fluids. The test sti^ comprises an elongated plastic pait inchidmg a hinged portion to allow a first portion to be folded over a second
portion. A series of layers of test strips_are disposed between the folded-over portions of the test strip. Hie step strip is configured such
that the chemistry layers are placed in contacting engagement with <me another, but not compressing one another. A reflectance photometer
Is provided and includes various feanires, includiiig a lot number reader wherein if the test strip does not match the memory module, a test
is not performed, and the user is instructed to msert a correct memory module.

FOR THE FURF05ES OF INFORMATIOS OSU

Codes used to identify States
applications under the PCX.

AT

Ausiria

AU

AianJia

BB

Barbados

BE

Belgium

BF

Bvfcina Fiso

BG

Bulgaria

El

Bcnm

BR

Bnzil

BY

Belarus

CA

Ctn«la

CF

Central Africa) Republic

CC

Congo

CH

Swiucrland

a

Cted*lvoire

CM

Cnneioui)

CN

Ouna

CS

Czccboftlovakia

CZ

Czech Repoblic

DB

Gomany

DK

Demnaric

ES

Spain

n

Finlnd

FR

Fnmce

GA

Gabon

party to the KT on the front pages

CB

United Kmgdom

CE

Ceotfia

CN

Guinea

GR

Greece

HU

Hungary

IE

Ireland

IT

lUly

JP

lapao

KB

Kenya

KG

KyifyMin

KF

Deinocntic People*! Republic

of Kofta

KR

Republic of Korea

KZ

Kazakhstan

U

Liecbtenstein

UC

Sri Lanka

Ul

LfnembQiug

LV

Laivia

MC

. Monaco

MD

RepobHcofMobtova

MG

Madagascar

ML

Mas

MN

MongpBa

pjmphtrt% puHi^hi!!^- mtrmalional

MR

Muiniattta

MW

Malawi

NE

Niger

NL

Netherlands

NO

Norway

NZ

New Zealand

PL

Poland

FT

romigal

RO

Rcanaaia

RU

RusUan FedentioR

SO

Sudan

SE

Sweden

SI

Stoveoia

SK

Stovakia

SN

Senegal

TD

Chad

TO

Togn

TJ

Ta^Kstaa

TT

Trinidad and Tobago

UA

Ukn^

US

United Stales of America

uz

Uibddstan

VN

ViclNam

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PCT/USW/12550

APPARATUS AND METHOD FOR DETER MTNIHG StlHSTAIf^ Ffff
CONTAINED IN A BODY FMITfl

BACKGROUND OF TflE INVENTION

The present Invention relates generally to an ass&y systen

for biological and nonbiological fluids » More particularly, the

present invention relates to an apparatus for separating serun

5 or plasma from particulate matter and then optoelectronically

evaluating the serum or plasma in order to measure analytes

vithin the serum.

It has long been desirable to utilize devices that can be

used for on-site testing of blood products. Particularly

10 important is the analysis of body fluids from humans and animals

to diagnose disease, .monitor the course of therapy, or determine

the presence of illicit drugs. . Commonly, the analytical methods

used to carry out these objects are , performed on blood samples.

Clinical chemists have a preference for working with serum

15 over plasma and plasma over whole blood because, of the clarity

of the sample matrix and the lack of interfering substances from

the solid portion of the blood. In order to facilitate

analysis, a separation step must be corrled out since the

presence of red blood cells, either intact or hcmolyzed

20 interferes with the signal generated hy thf» chemical reaction

performed by the test*

Conventionally, the separation of blood components has been

carried out by placing a blood sample in a centrifuge and

cehtrifuging the sample for ten minutes at approximately 3,000

25 rpms. The serum obtained from this centrifuging step is then

used to carry out the test, thus avoiding interferences from .

blood solids such as red blood cells.

An embodiment for chemical tests called dry reagent strips

was developed first for urinalysis. Thereafter, various efforts

30 to combine dry reagent strip technology in blood ^testing were

started in the early i950*s. Notably, U.S. Patent No. 3,092,465

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discloses a reagent in a bibulous carrier with a super imposed
semipermeable coating to exclude the chemical and nonchemical
interferences from red blood cells. The device, while
performing analysis on whole blood, still required additional
manipulations by the user, in the form of washing of excess
bipod after a specified time interval. Additionally, U.S.
Patent Nos. 3,552,925 and 3,552,928 disclose the use of salts
and amino acids to perform In-sltu separation. U.S. Patent Ho.
4,477,575 discloses the use of a glass fiber matrix.

More recently, membranes have been employed in a variety of
devices. These Include devices disclosed in the following
United States and foreign patents and publications: U.S. Patent
HQS. 4,774,192 and 5,166,051; European Published Applications EP
0408222 Al, EP 0408223 Al, EP 0407800 A2 and EP 0388782; and PCT
Published Applications Nos. WO 93/22453 and WO 90/10869. The
use of the various membranes disclosed in the above patent
documents operate on size exclusion principles, and several of
these ire limited by rates of capillary flow and do not
completely, eliminate interference from Intact or hemolyted red
blood cells. Fresh red blood cells are elastic In nature and
may pass through pores smaller than their nominal diameter.
Hemolysis may occur on contact with some of the architectural or
chemical components of the strips. Consequently, errors may ba
introduced into the measurement system.

U.S. Patent No. 5,104,619 discloses a disposable diagnostic
system comprising a test card having a substantially flat body
and a generally cylindrical reagent pad pocket formed in a
central area of the flat body. A reagent chemistry pad is
disposed in the pocket and a snap fit cover is received in the
30 pocket and arranged over the pad to retain the pad In position*

the device size and configuration allows for bar code graphics
to be printed on the underneath side of the device. The bar
code may contain lot specified data about the reagent chemistry »

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and ie read by the meter during device insertion. This data may
further contain critical parameters for the software algorithm
within the meter electronics.. U.S. Patent No. 5,13.9,685 also
discloses a separation filter assembly having a snap fit lid.
5 In this patent, glass fibers are utilized and maintained in a

compressed state under pressure.

Accordingly, a need exists for an integrated system for
assaying analytes and whole blood samples which are not affected
by the chemical or physical interferences normally caused by red
10 blood cells and other portions of whole blood.

SUMMARY OF THE IWVEWTTQW
The present invention, in one form thereof, comprises a dry
solid phase diagnostic test strip and system for the chemical,
enzymatic, and/or immunological analysis of whole blood
15 analytes, comprising a reflectance photometer, a solid support

strip, a porous detection zone member, a permeable spreading
layer, an overlay sample receiving membrane containing an agent
for the exclusion of intact red blood cells and a strips-
receiving platform for positioning the strip inside the
20 reflectance photometer. The detection area membrane may contain

chemical, enzymatic, and/ or immunological reagents that generate
. specific signals in the presence of a target analyte. The
agent, in contact with the overlay membrane, prevents passage
and hemolysis of red blood cells while facilitating rapid
25 transport and reaction of the plasma or serum portion of

^introduced whole blood samples.

In addition, the present invention, in one form thereof,
comprises a reflectance photometer which utilizes test strips
that are color coded for test differentiation. For example, a
30 blue strip may indicate a glucose test, whereas a red strip may

indicate a cholesterol test. These colors are then divided into
shades such as 64 shades of blue egual to 64 lot numbers of
.glucose strips. The photometer includes a separate optical read

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head that determines the color and shade of the base of the test
strip device as the strip is inserted into the photometric
Instrument. The shade is converted into a lot number ranging
from 1 to 64. The instrument also has a memory module
(preferably an electrically erasable programmable read-only
memory) that has a corresponding lot number to the shade of the
strip to ensure lot number verification. The instrument then
compares the inserted memory module programmed lot number to
ensure that it is the same lot number as the test strip, if the
strip lot number does not match the memory module lot number,
the test is not performed, and the user is instructed to insert
the correct memory module.

The lot number verification allows for the automated coding
of lot numbers so that the user does not need to enter a lot
code for each vial of strips. This prevents the running of the
incorrect, old, or expired lot number tests in the instrument.

The -plug-in memory" of the module Includes the lot number
of the test strip, the expiration date, and the performance
criteria foi: the actual strip measurement. The performance
criteria include the wavelength, measurement algorithm, ond
unreacted density qualifications necessary for a valid teat
result.

The optoelectronic measurements of the chemistry test
reaction on and in a surface enhances the dynamic range of the
dry phase test. Algorithms that read at different wavelengths
at different times in the chemistry reaction can extend the
dynamic range of the test system. This Is particularly
applicable when using multiple chromophores in a single
measurement system. The early portion of a chemistry could be
read at the peak wavelength of a reaction, while the later
portion or darker or more dense portion of color development
could be read at a wavelength not near the peak of the color
development. In addition, different chromophores may respond In

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a wore linear manner in different portions of the dynamic range
of the chemistry. Manipulation of these two data points can
Bignif icantly increase the dynamic range (in mg/dl) of a
chemistry reaction.
5 The optoelectronic measurement of the chemistry test

reaction on and in a surface reduces error due to orientation of
the surface to be read to the instrument. Multiple wavelengths
and different angles are used to correct possible problems in
positioning the strip in the instrument. If the detector is at
"0" angle and the emitters of the same or different wavelengths
are at different angles (e.g., one at 40» and one at 50*}, the
tilting of a surface will positively contribute to one reading
while it will contribute in a negative manner to the other
reading thus it is able to cancel the error presented by the
15 angle presentation of the surface. These same measurement

methods can be used to eliminate interferences from substances
such as bilirubin and others.

The optoelectronic measurements of the chemistry test
reaction on ,and in the surface enhance the stability of timed
20 and untimed dry phase chemistry reaction. Algorithms are used

to determine the "end point" of a chemistry. In other words,
measurements can be done at similar or dissimilar wavelengths to
predict the stable portion or end point of a chemistry. If
kinetic measurements are made, the kinetic readings can be
25 subjected to an algorithm to determine that the rate is slow

enough to declare the extrapolate chemistry is at an end or
completion. When known standards are run and predicted by this
pseudo-endpoint, the same measuring criteria can be applied to
unknowns to determine the "endpoint" of the test reaction.
30 The use of colored or shaded visual indicators in the

instrument enhance the interpretation of test results. A
colored bar graph is used to aid the user in knowing when the
user test results are in a normal or safe range. Out of range

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WOW13707 PCTAIS9S/12550

colore such as orange for caution and red for danger are used
when results are outside the green "safe" range. This is
particularly useful to new testers who are not familiar with the
number scale of the different test results, a voice module can
5 also be used to warn the user of unsafe results or operation of

the Instrument system to make the system usable by the visually
Impaired by providing, for example, a sound beep for each unit
of glucose during a glucose test.

PRIEF DESCRIPTTOtf pp TUB nnj^y ry||f^c^
Fig. 1 is a perspective view of the reflectance photometer
in accordance with an embodiment of the present invention;

Pig. 2 is an exploded perspective view of the plastic test
strip of present invention in its unlocked position;

Pig. 3 is a perspective view of the plastic strip of rig. 2
15 in its locked position; and

Pig. 4 is a sectional view of the plastic strip;
Pig. 5 is a block diagram schematic of one embodiment of
the reflectance photometer of the present invention;

Pig. 6 jis a graph plotting sample size, elapsed test time
and percentage of reflectance illustrating how endpolnt
determinations may be utilized to speed chemistry measurement.

DESCRIPTION OF TH E PREFERnED EMBODIMEMT
In accordance with the embodiment of the present invention,
the diagnostic chemistry measurement device 10 for dry solid
25 phase chemical, enzymatic. Immunological assay of whole blood or

sera analytes is made up of an Injection molded carrier test
strip 20 In which several porous and nonporous materials
containing chemicals and reactants are contained for the purpose
of generating a detectable signal In the presence of certain
analytes. The test strip 12 Is inserted Into a reflectance
photometer. The reaction material layer on the test strip 12 la
held in intimate noncompressed contact with a whole blood
separation layer In the absence of adheslves for the purpose of

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providing a liquid sample free of red blood cells to the
reaction layer or layers.

HQM^r

The holder test strip 12 of this invention acts as holder
5 for the different layers of the test reaction system. It

provides a convenient handle as well as a mechanism for placing
test strip 12 into an instrument 10 for the reading of the
density changes of the reaction layers. As ehown in Pig. 2 test
strip 12 includes an elongate body 16 preferably formed by
10 injection molding. Elongated body 16 includes a first end

portion 18 and a second end portion 20. A hinged portion 22 is
located between first and second and end portions 18 and 20 so
that first end 10 is foldable over elongated body 16 into
contact with second end 20.
^5 As shown in Fig. 2. first end portion 18 includes a opening

24 while second end portion 20 includes a complementary spaced
opening 26. When first end portion IB is folded over body 16,
each opening 24 and 26 are aligned. In its folded position as
shown in Pig. 2 opening 24 in test strip 12 defines an area for
20 depositing a body fluid sample while opening 26 defines an area

in which optoelectronic measurements of chemistry test reactions
are conducted.

Test strip 12 further includes an adhesivelesB carrier
'layer 14 formed from, for example, three particular layers. In
25 a standard diagnostic test strip, carrier layer 14 may include a

disbursement layer 28, formed of for example woven materials
such as. polyester or cotton, for rapid and even disbursement. of
body fluid along carrier layer 14. Beneath that may be included
a separating layer 30 constructed of known materials such as
30 shown in Table IX infra, that, when exposed to a sample liquid,

may separate analyte and analyte disrupting elements such as red
blood cells from whole blood. This action would permit the
serum analytes to pass through separating layer 30 while

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preventing red blood cellB or other analyte disrupting elemente
from passing through. The last layer shown in rig. 2 is that of
the test reaction membrane 32 on which the dry chemicals and
reactants are contained for generating a visible signal in the
presence of serum analytes. Molded carrier body 16 serves as a
support for the reacting and nonreacting layers 28, 3o and 32
which may be formed from papers, membranes and deles materials.

The test strip holder 12 positions the different layer
materials 28, 3i, 32 within the holder the correct x, y, and Z
axis positions. Carrier layer 14 made up, for example, the
disbursement separating and test reaction layers 28, 30 and 32
are held in noncompressed adhesiveless locations by first end
portion IB folding over to second end portion 20. This may be
accomplished in a number of different ways. The preferred way
of noncompressingly holding carrier layer is of an upstanding
annular rim 34 may help locate the carrier layer 14 within test
strip 12. Additionally, small upstanding protuberances 36 along
second end portion 20, radially located away from opening 26
prevent movement of carrier layer 14. The purpose of both
annular rim 34 and small upstanding protuberances 36 is to hold
the layers of carrier layer 14 without compression between
opening 24 and opening 26, thereby preventing pooling of any
sample within carrier layer 14. This consideration of
noncompresslon of the carrier layer 14 is of greater importance
25 when larger numbers of layers are utilized. The positioning of

a carrier layer 14 without adheslves or compression allows for
efficient transport of sample and reactants contained in the
system and test strip 12. Annular rim 34 or alternatively other
areas of test strip 12 may Include sawtooth protrusions to
increase flow rate through carrier layer 14

Test strip 12 includes a locking mechanism to prevent any
unlocking of front end portion 18 from its folded position over
elongated body 16. As shown in Fig. 2, one type of locking

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nechanisn may include a plurality of upwardly extending tabs or
projections 38 that interfit or lock into corresponding openings
40 in first end portion 18. When first end portion 18 is folded
to second end portion 20, locK projections 38 will interfit and
5 snap lock within openings 40. Other types of one way locking

mechanisms may also be used, such as snap rivets.

More than one test reaction system can be housed in a teat
strip 12. A second set of holes 24, and 26 may be included in
test strip 12 so that two testa may be run at once.

The described holding mechanism allows for the rapid
separation of whole blood into its liquid and solid components.
It also allows sample volumes as low 2.0 microliters to be used
in dry phase chemistry reactions. Test strip 12 allows the use
of several reaction and non-reaction layers. A typical holder
15 could contain from 1 to 8 layers of material with thicknesses

from approximately 0.002 inches to 0.007 inches, for example.

Chemicals and materials are employed to allow for the
treatment of samples such as whole blood, which will allow the
whole blood sample to be separated without disrupting the red
20 blood cells while rapidly moving the liquid portion of the whole

blood sample to one or more reaction sites in the holder,
normally on a test reaction membrane 32. These chemicals can be
composed of polymeric and nonpolymeric substances that are dried
onto, one or. more surfaces of the materials contained in the
25 device holder.. Additionally, light metal salts of elements such

as Potassium, Lithium, Sodium, and Calcium may be utilized to
treat red blood cells before and during the separation process.
The materials which may be used in the holder for treatment by
or containment of these chemicals can. be composed of woven,
30 nonwoven, napped, or flocked materials.

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Analvtc.i

A wide variety of analytes can be detemined by using the
disclosed apparatus. Examples are given in tables I and li.

Further, given the small size and robust nature of the
reagent strips and Instrumentation, analyses need not be limited
to traditional clinical laboratory settings. The device of the
present Invention is also simple enough to be used by people
vlth minimal or no chemical or medical technology training.
This advantage allows use at home, or by mobile health care
delivery people. Examples of this are diabetics that must
monitor themselves for glucose and ketone bodies, patients on
home dialysis who would benefit by monitoring of urea nitrogen
and people endeavoring to lower their cholesterol levels.

Further, by combining several different reagents on a
single support, a panel of tests may be done. Examples of this
would be a liver panel consisting of ALT, AST, Alkaline
Phosphates. A diabetic panel might consist of glucose, beta
hydroxybutryrate and glycated hemoglobin. A coagulation panel
might consist of Prothrombin time, ACTT, and ACT.

FAMILIES OF ANALYTES BY STRUCTURE

Family Examples

Carbohydrate

glucose, lactose, galactose

Nitrogen Moiety

urea nitrogen, creatinine/ uric acid

Lipid

cholesterol, triglycerides, LDL, HDL

Enzyme

ALT, AST, Alkaline Phosphatase,. CPK, CK«*KB

Hormone

HOG, UI

Therapeutic Drugs

theophylline

Drugs of abuse

cocaine, marijuana, barbiturates,
salicylates

Electrolyte

Na*, K*, Cl", Li^, 00^

Nucleic Acids

infectious disease, forensic applications,
genetic disorders

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FAMILIES OF AMALYTBS BY DISEASE

Table TT

Dleease Examples

Diabetes

glucose, beta hydroxy butyrate, hemoglobin
^Ic

Liver problems

ALT, AST, bilirubin

Acidosis/Alkalosi

po^, pCOj, pll

Hypertension

nutritional status

Ca , Hg**, Zn**, trace minerals

Examples

The following illustrative examples teach various
combinations of buffers, dyes, stabilizers and other reactive
and functional components which may be combined by a person
having ordinary skill in the art into the system test reaction
areas.

Table IX gives various types of dyes and indicators used in
diagnostic reagents.

Example /I

Glucose measuring system
TABLE III

Ingredient

Function

Amount

Available from .

Glucose oxidase

reactant

25,000

Sigma Chemicals,
St. Louis, MO

Peroxidase

reactant

75,000

Sigma Chemicals,
St. Louis, MO

Silwet 7500

surfactant

0,10ml

Dow-Coming, ^
Midland, HI

PVP K 30

ensyme .
stab.lliser

O.SOgms

ISP, Linden, NJ

Citric Acid

Buffer
system

1 • 25gm8

Aldrich Chemical,
Milwaukee, HI

Sodium citrate

Buffer
system

O.XOml

Dow^Corning,
Midland, MI

DOW 1520

ant i foam

1 . OOgms

Aldrich Chemical,
Milwaukee, HI

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A AAP

chroinophore

0.25gms

Aldrich Chemical,
Milwaukee, WI

3,5 DCHBS

chromophore

0 .2511193

Boehringer Mannheim

Distilled H^O

solvent

QS to loonl

Preparation: Approximately soul of distilled ir^o was placed In
a beaker on a stirring plate. A magnetic bar was added and the
Ingredients added sequentially after the previous gradient was
dissolved and dispersed. After all Ingredients were added the
volume was adjusted to looml 6t distilled lljO.

10

Example 2

Triglycerides measuring system

TRIGLYCERIDES + H,0 tWmt*.^ tWB«ff CLVCEROI. * FREE FATTY ACIDS
GLYCEROL + ATP SUSISUUSSa ^ . _ GLYCEROPHOSPHATE + H,0,

HjO» + 4-AHIMOANTIPYRINE + DCHBS BSSSiim QUINOMEIMINE CHROMOPHORE

TABLE TY

Ingredient

Function Amount

Cholesterol esterase

reactant

15,000 units

Shinko*American,

M» y • , He y •

glycerol kinase

reactant

5,000 units

Shinko-American,
M.y., M.y.

glycerophosphate
oxidase

reactant

5,000 units

Sh i nko-Amer i ca n ,
N* y • , He y . .

peroxidase

reactant

5,000 units

Shinko-American,
Y» , H* y •

4 AAP

chromogen

l»00gm

Aldrich

3r 5 DCIIDS

chromogen

0.25gm

Boehringer
Mannheim

MBS

buffer

2e50giii

Research
Organics

PVP K30

stabiliser

O.SOgn

isp

glucose

filler

2e56gm

Sigma

triton x-loo

surfactant

O.iogm

Boehringer
Mannheim

Distilled II^O

solvent

Q5 to lOOml

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Preparation: Same as example #1
Example 3

Cholesterol measuring system (all amounts approximate)
TABLE V

Ingredient Function Amount Available from

Cholesterol Oxidase

reactant

10,000

Shinko-American,

cholesterol esterase

reactant

7,000

Shinko-American,

sodium phosphate
0.5 M pll 7.0

buffer

750 ml

Dow*corning,

B. S« A.

surfactant

15 gm

Aldrich Chemical^

peroxidase

reactant

170,000

Sh i nko- Amer i can /

DOSS

surfactant

7.0 gms

Doehringer
Mannheim

sucrose

stabilizer

1,0 gms

Sigma Chemicals,

TMB

chromogen

10.0 gms

Aldrich Chemical,

Distilled II^O

solvent

QS to 100 ml

Preparation: same as example fl

Alternatively, the chroihogen may be prepared in an organic
solvent matrix and treated as a first or 2nd application to the
membrane or paper.

TABLE VI

Ingredient

Function

Amount

Available from

Acetone/methanol

i:l

solvent

100 ml

Aldrich

Tetramethyl
benezidine

solvent
chromogen

1. OOgm

Biosynth Inc. ,
Chicago, IL

Example 4

Blood Urea Nitrogen Measuring System

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TABLE YIT

Ingredient

Function

Urease

Ireactant

H,0

solvent

Bconthynol blue

chroittogen

PVP K90

film former

PructoBe

filler

Preparation: Same ae experiment /i.

Table VTT7

Types of Tndicatnrn
Chromogenic substrate
Redox

Leuco dyes

Oxidative couplers

Behzidene Derivatives
Fluorescent labels
Dye releasing system

Table ix

Separation mechanisms used in dry reagents

Chemical

Physical

Mechanical

Dextran

hydrophilic polymers

centrifuge

sugars

porous latex films

filters

lectin

polymer 6 swelling agent

filters ft pressure

amino acids

membranes

membranes ft

differential

pressure

PE6/polyacrylate

microf iber cloth

wedge shape

thrombin

napped cloth

gels

sintered porous matrix

coagulants

density gradient

agglutinating
agents

glass fibers

1 1

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amine polymers

hollow fibers

trivalent cations

ineinbrane i

' — -i

Spectrophotometer

The present invention also includes use of a
spectrophotometric device 10 for determining the density of the
color reaction on and in the membrane surface of the test
5 reaction layer 32 within test strip 12. Photometric device 10

as shown in Fig. 1 Includes a hand-held housing 50 for
containing electronic control circuitry for operating the
aforementioned tests. In the embodiment shown in Fig, i, a test
strip holding region 52 is located above three light detectors
10 or sensors 54 each disposed within a port 56. During test

operation, a. test strip 12 is inserted into holding region 52 so
that test strip openings 26 are located adjacent ports 56.
Light sensors may take a reading from light reflected from the
exposed test reaction membrane layer 3 2 or from test strip 12
^5 itself to determine its color.

Housing 50 further includes a specialized display device,
such as a liquid crystal display 56. Display 58 is utilized for
relating test results and other information to the user^ In
particular/ a color scale 60 is used to facilitate
20 interpretation of test results operating concurrently with

digital display segments 62. Additional display segments on
display 56 include a test wait indicator segment 64 to inform
the user to wait while device 10 is performing the selected
tests, and a test name segment 66 which the unit determined from
25 the type of test strip 12 inserted.

Color scale 60 may easily by. constructed by a plurality of
shaded or colored segments arranged adjacent each other to form
a bar graph like indicator. Electrically controllable segments
6B are oriented over the color or shaded segments so that when
30 segments 68 are activated segments 68 become dark, preventing

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certain colored or shaded eegmente 60 fro» being visualized or
viewed, segments 68 that are not activated permit the
underlying colored or shaded segments of color scale 6b to be
visualized, m this way it is possible for an electronic
> control to permit only a single colored or shaded segment to be

viewed thereby communicating test results.

A possible result range spectrum for cblor scale indication
segments may include particular colors with particular teat
result , meanings such as:

Very high result danger, RED
high result danger, red
high result caution, YELLOW
high result caution, YELLOW
high normal ic^esult, GREEN
normal result, GREEK
normal result, GREEN
low normal result, GREEN
low result caution, YELLOW
low result caution, YELLOW
very low result danger, RED
Color scale 60 permits an unsophisticated user to instantly
visually determine, in one embodiment, if a test result is
normal (a green segment visualized), slightly abnormal (a yellow
segment yisualized) or dangerous high or low result (a red
segment visualized). Alternatively, if a color liquid crystal
display is utilized, the electronic control for test unit lo nay
directly indicate a colored segment, rather than covering all
but one colored segment.

A suitable Instrument, such ds a diffuse reflectance
spectrophotometer 10 with appropriate software, can be made to
automatically read reflectance at certain points in time,
calculate the rate of reflectance change, and by using
calibration factors and software, output the level of analyte in

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the fluid tested. The electronic control wechanisw of
photometric unit 10 is shown in schematic form in Fig» 5. one
or more light sources 70, for example high intensity light
emitting diodes (LED) are disposed in housing 50 to illuminate
5 test strip 12 as shown by arrows 72, A light detector or sensor

54, for example a photo transistor, is able to take a reading of
light reflected either from the surface of test strip 12 or from
its associated test reaction membrane 32. Light source 70 and
light sensor 54 can be adapted to generate or respond to
10 particular wavelengths of light.

Sensor 70 transmits a signal to an amplifier 74 as is known
in the art. One type of amplifier available for use is, for
example, a linear integrated circuit which converts the
phototransistor current to a voltage signal*

Appropriate electronic circuitry is utilized to take the
output of amplifier 74, normally a sample and hold unit 76, and
transfer the signal to an analog-to-digital converter 78.
Analog-to-digital converter takes the analog voltage output from
the sample and hold unit 76 and converts it to, for example a 16
20 bit binary digital number upon command of a microprocessor/

microcontroller unit 80.

Preferably an electronic microprocessor/microcontroller 80
Utilizing digital integrated circuitry is used to time selected
tests, read signals, and together with associated programs and
5 data memory 82, calculate and store reflectivity valves and

calculate analyte levels from the stored data. .

Additional information for particular tests nay be stored
in a removable EEPROM unit 84 operably connected to
microprocessor/microcontroller 80. EEPROM unit 84 is an
0 interchangeable plug-in memory module containing measurement

parameters, software, calibration data, and reagent recognition
data for particular test strips 12. Additionally, EEPROM unit
84 contains the shelf life data and identity verification

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)

information for particular production runs or lots of test
strips 12.

Automated lot coding is done by the color coding the
Plastic material used to make the test strip holder 12, The
color used in test strip holder 12 preferably has 16 different
densities that can be distinguished by at least one of the
vavelengths used in the optical sensor head 54 of instrument 10.
For instance the dynamic range of the I reflectances of the
strip holder color could be as follows to determine the
different shades of color density:

%Reflectance iReflectance Lot /

Green LED Red LED

70
65
55
50
45
40
35
30

70
65
60

1

2
3
4

5
6
■ 7
8
9
10
11

55 12

50
45
40

13
14

15

35 16
AS the strips 12 are inserted into device lo, the instrument
detects a change in the measurement area. This change indicates
that a strip 12 has been inserted into the instrument 10. As
the instrument detects the insertion of a test strip 12, it
reads the densities of at least one df the LED -s and calculates
the lot number by the above table. Instrument lb then goes to
the EEPROM port connected to microprocessor / microcontroller 80
which has an BEPROM unit 84 inserted. Instrument 10 checks to
see that the .EEPROM preselected lot number is the same as lot
number of test strip 12 that had been inserted into the
instrument. If the lot numbers are the same for test strip 12
and EEPnoM 84, the instrument downloads the information
contained in the EEPROM and proceeds with the test analysis^

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The instrument 10 reads the density of the unreacted strip to
assure quality of the strip before the test is initiated, if
quality is passed then the instrument instructs the user to
apply a sample.

5 A sample is then applied and instrument 10 begins a

measurement cycle to ensure that the proper amount of sample vai
applied to the test strip. When the instrument has determined
that enough sample has been applied, it then goes Into another
cycle to measure the end of the chemistry reaction. When the
10 end of the chemistry reaction has occurred, then the instrument

measures the final density and compares it to a measurement
algorithm stored in EEPRON unit 84. This measurement algorithm
then determines the concentration of the test to be measured by
comparing the measured density (darkness) of the color formed
15 and comparing this density number to a table of values through

the use of an algorithm stored in the EEPROM unit 84.

After a particular test strip is selected and placed in the
unity a sample, normally a whole blood sample from a fingertip
or from a plpiter tip (which could have gotten Its sample from a
20 tube of blood as in a laboratory type situation) Is applied to

the sample application spot, opening 24, on test strip 12. A
dispersement layer 28 causes the sample to quickly spread over
the entire area of carrier layer 14. The separation layer 30 of
the test strip spot is allowed to separate out the soidds (red
25 blood cells and other analyte disrupting elements) from the

liquid (plasma or sera or other analyte containing portion).
The separated fluid, i.e the plasma, sera, or other analyte
containing portions, moves to the test reaction membrane layer
32 below the separation membrane 30. The above fluid migration
30 causes the reactants (analytes such as glucose) in the sample to

come into contact with the reactants in test reaction membrane
layer 32 »

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Analytes/fluld contacts reagent layer reaction 32 and
Initiates an appearance or disappearance of color, depending on
its particular reaction. The above presentation of analyto to
the reaction layer 32 causes the desired reaction to occur.
This reaction causes a color change that can be detected both
vlaually and by the instrument. The color change is then
converted into a digital result on the instrument LCD as
described above, a comparison color chart can be used to
visually determine a reaction quantity scale as in litmus paper.

instrument lo can use different wavelengths at different
density portions of the reaction to maximize the dynamic range
of the chemistry and the limits of the Instrument at a
particular wavelength.

The "end-point- of the reaction is defined as a point where
there appears to be no change or a very small change in density.
That is, the chemistry changes color proportional to the
concentration of the reactance that has come into contact with
the reactance materials in the test pad (membrane) . This small
amount of change can be a change per time period. An example
would be as per the graph in Fig. 6. Detailed Information used
to generate this graph is that the changes per 5 second time
period during the beginning of the test reaction would be
greater than 5% reflectance per 5 second time period. When this
change is less than 1% reflectance per time period it can be
said that the reaction is complete or at an endpoint. The
instrument stores this percentage reflectance at this time and
uses as above to determine the concentration of the analyte
tested for in the test strip.

The Kubelka-MonK ecjuatlon of K/S-(I«-ref lectance)^ divided
by (2 X reflectance) can be Used to linearise the percentage
reflectance values. This linearization simplifies the algorithm
necessary to calculate results. This pseudo endpoint chemistry
allows a more stable read time, which in turn allows for a more

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reproducible answer. Pseudo endpoints also permit a more rapid
assay to be performed. Certain other glucose monitoring systems
incorporate pre-deterinined timing circuit. This pseudo ehdpoint
allows for a different method to be used in measuring chemistry
5 reactions, provided one can determine the endpoint of the

chemistry by a method other than timing.

Multiple wavelengths are used to enhance the dynamic range
of a chemistry. This is particularly useful when one uses a
multiple chromophore indicator system as do some of the above
10 mentioned chemistries. Early portions or low concentrations of

a test such as glucose can use a broad range indicator such as
TMB to increase sensitivity in the low to mid range of the
chemistry. When the test concentration is higher or the
reaction faster, a different chromophore is focused upon to
15 determine more dynamic range than the previous chromophore.

This allows one to expand the dynamic range by two different
methods •

One can also use wavelengths on the peak for more dynamic
range and wavelengths off the "peak" absorbance of the test

20 system to enhance or reduce dynamic range and also to enhance or

reduce the "pseudo endpoint" algorithms. Manipulation 6f these
four factors, chromophore A, . chromophore 0, wavelength 1 and
wavelength 2 can allow one to better define the "pseudo end*
point" algorithm and also allow one to optimize the dynamic

25 range of the chemistry which in turn allows for increased

sensitivity throughout the chemistry reaction range with greater
precision.

Multiple wavelengths can also be used with different angles

I . . .

of emission to correct possible problems in positioning the
30 strip in the instrument. If the detector is at "0" angle and

the emitters of the same or different wavelengths are at
different angels (one at 40* and one at 50*) the tilting of a
surface will positively contribute to one reading while the

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10

other contributes In a negative manner thus cancelling the error
presented by the angle presentation of the surface. These saioe
measurements methods can be used to eliminate interferences from
substances such as bilirubin and others, when the angle of
light incidence is increased from Improper positioning of a
chemistry read surface to the instrument optics, errors of both
gloss and angularity are introduced into the measuring system
and can give false low readings.

Exampl^iy

1- Indicators and chromogens advantageously used in combination

a. wide range pli test

Dromothymol blue and methyl red covers pll range of 5

through 9

b. 4 amino antipyrine +3,5 dichlorohydroxybenzene
15 sulfonate (4AAP+3,5 DCHBS)

c. TMB4Chromotropic acid

d. Syringaldazine + Vanillin Azine

2. Color coding for test and lot identification

a. blues, 16 different shades (density)

b. reds, 16 different shades (density)

c. greens, 16 different shades (density)

d. yellows, 16 different shades (density)

e. oranges, 16 different shades (density)
browns, 16 different shades (density)

g. magentas, 16 different shades (density)

h. light blues, 16 different shades (density)
light reds, 16 different shades (density)

j. light greens, 16 different shades (density)

k. light browns, 16 different shades (density)

1. light magentas, 16 different shades (density)

m. cyan, 16 different shades (density)

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n* light cyan, 16 different shades (density)

It will be appreciated that the foregoing is presented by
way of illustration only, and not by way of any limitation, and
that various alternatives and modifications may be nade to the
illustrated embodiment without departing from the spirit and
scope of the invention.

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10

10

WHAT IS CLAIHED IS:

1. A diagnostic test strip for use In an analyzer for
»easurlng analyte In a sample, said test strip comprising:

an elongate body Including a first end, a second end,
and a hinged portion between said first and second ends, said '
first end being foldable over said body, said first end and said
second end each having an opening that are aligned when said
first end Is folded; and

an adheslveless carrier layer disposed without
compression between said first end and said body whereby sample
communicated to said carrier layer Is prevented from pooling
within said carrier layer.

2. The test strip of Claim 1 in which said carrier layer
includes a separating layer that when exposed to a whole blood
sample excludes red blood cells from passing therethrough while
allowing the liquid portion of the whole blood sample to pass
therethrough.

3. The test strip of Claim l having a carrier layer
utilizing samples in the range of 2. Qui to 10. Qui to generate a
reaction to accurately test a selected analyte.

4- The test strip of Claim 1 in which said carrier layer
includes:

a separating layer that when exposed to a sample
liquid having both analytes and analyte disrupting elements said
separating layer excludes said analyte disrupting elements from
passing therethrough while allowing the analyte portion of the
sample liquid to pass therethrough; and

a test reaction membrane adjacent said separating
layer that creates a gradient color dependant on the
concentration of selected analytes in the analyte portion that
had passed through said separating layer.

5. The test strip of Claim X in which said carrier layer

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includes a spreading layer over said separating layer to cause
sanple to substantially evenly enter said separating layer.

6. The test strip of Claim 1 in which said carrier layer
may test more than one analyte at one time.

?• The test strip of Claim 1 in which said body includes
a locking means lock together said first end and said body,

B. A diagnostic test strip for use in an analyzer for
measuring analyte in a sample, said test strip comprising:

an elongate body including a first end, a second end,
and a hinged portion between said first and second ends, said
5 first end being foldable over said body, said first end and said

second end each having an opening that are aligned when said
first end is folded;

an adhesiveless carrier layer disposed between said
first end and said body of said carrier layer whereby the
10 accuracy of measured ana ly tea is increased; and

non-'compressive means' holding said carrier layer in
place between said first end and said body whereby pooling of
sample within said carrier is prevented,

9; The test strip of Claim e in which said non-
compressive means comprise protrusions to locate said carrier
layer in place whereby said carrier layer is maintained in Known
locations along the X, Y and Z axis.

10. The test strip of Claim 8 in which said non-
compressivie means comprise sawtooth protrusions to locate said
carrier layer in place whereby said carrier layer is maintained
in known locations along the X, Y and Z axis.

11. The test strip of Claim 8 in which said separation
layer is treated with light metal salts to reduce red blood
colls in the sample.

12. The test strip of Claim 8 in which said first end
folds over said carrier layer and locks to said body causing the
layers, of said carrier layer to be in adjacent contact without

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adheslves or oon-presslon whereby efficient seperatlon of red
Wood cells from plasma In whole blood samples.

13. The test strip of claim x further comprising a tab and
an opening one of which on said first end the other on said
second end so that when said first end folds into contact with
•aid body, said tab Interfits with said opening to lock said
first end with said body.

14. A chenistry measurement system comprising:
a test Instrument with a light source and light

sensor;

diagnostic test strip for use in analyzing a sample,
an elongate body including a first end. a second end, and a

hinged portion between said first and second ends, said first

and being foldable over said body, said first end and said

second end each having an opening that are aligned when said

first end is folded, said test strip having an adheslvelese

carrier layer disposed without compression between said first

end and said body, said opening adapted to receive said sample,

an electronic control for computing particular test

results on light Incident on said light sensor that was

reflected from said testi strip; and

display means for displaying said test results.

15. The measurement system of Claim 14 In which a "
plurality of test strips are utillEed for particular chemical
tests, said test strips color coded for identification of which
said particular chemical test said test atrip is operable.

16. The measurement system of claim 15 in which a
plurality of test strips are utilized for particular chemical
tests, said test strips color coded for identification of said
particular chemical test for which said test strip is operable
and lot designator, said light sensor sensing said color of said
test strip when said strip is inserted into said test instrument
and sending a coded signal to said electronic control, said

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10

10

15

20

electronic control determining the type of test and lot
designator of Inserted test strip and denying test operation
when said lot designator Is not within predefined limits.

17. A chemistry measurement system of claim 16 In which
said system tests for more than one analyte with a single test
strip.

18. A chemistry measurement system comprising:

a test Instrument with a light source and light

sensor;

a color cbded diagnostic test strip for use In
analyzing a sample, said strip having an elongate body including
a first end, a second end, and a hinged portion between said
first and second ends, said first end being foldable over said
body, said first end and said second end each having an opening
that are aligned when said first end Is folded, said test strip
having an carrier layer disposed without compression between
said first end and said body, said first end opening adapted to
receive said samplef

an electronic control for computing particular test
results on light incident on said light sensor that was
reflected from said test strip, said control determining from
said color of said test strip If said test strip is from a
particular production lot, said control operating said test,
operation only if said test strip is from a preselected
production lot; and

display means for displaying said test results if said
test is conducted.

19. A chemistry measurement system comprising t

a test instrument with a light source and light
sensor, said light source emitting light at multiple angles and
multiple wavelengths;

a diagnostic test strip for use In analyzing a sample,
said test strip comprising an elongate body including a first

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10

10

end, a second end, and a hinged portion between said first and
second ends, said first end being foidable over said body, said
test strip having an adhesiveless carrier layer disposed without
compression between said first end and said body;

an electronic control for conputlng particular test
results on light Incident on said light sensor that was
reflected from said test strip; and

display means for displaying said test results.

20. The chemistry measurement system of Claim 19 in which
said electronic control includes a removable erasable
programmable read only memory unit containing lot number data
and expiration data for particularly chemical tests.

21. A liquid crystal display matrix for a hand-held

chemistry measuring system, said display matrix comprising:

a display screen having a plurality of shaded segments
arranged adjacent each other;

a plurality of controllable segments disposed within
said screen oriented over said shaded segments, said
controllable segments preventing visualization of said shaded
segments when activated and permitting visualization of said
shaded segments when deactivated whereby results from said
chemistry measuring system are communicated by visualizing
selected shaded segments.

22. The liquid crystal display of Claim 21 in which said
shaded segments are aligned in a line forming a substantial bar
graph indicator.

23. The liquid crystal display of Claim 21 in which said
shaded segments are colored to indicate selected results from
said blood chemistry measuring system.

24. A method of testing analyte in a sai^le comprising the
steps of t

providing a chemistry measurement system having a
light sensor, test display, colored diagnostic test strips for

28

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use in analyzing a sample, each said test strip having an
adhcsiveless carrier layer attached without compression to said
test strip;

selecting a test strip of a predetermined color for a
predetermined test;

depositing a test sample on the carrier layer of the
selected test strip;

inserting said test strip into said measurement

system;

operating said measurement system to conduct the
predetermined test; and

displaying test results on said test display.

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PCT/US95/1255a

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1^

9*

>

or

od D

UI

21

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PROGRAM
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-J 1

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6/5

ANY REFERENCE TO
FIGURE 6 SHALL BE
CONSIDERED NON-EXISTENT
(See Article 14(2))